A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450 BM3
Open accessAbstract
Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.
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The approach outlined in Scheme 13 has succeeded in producing high levels of amorpha-4,11-diene from E. coli and artemisinic acid from S. cerevisiae in quantities exceeding those from A. annua itself (100 mg-1 g L-1).
“The approach outlined in Scheme 13 has succeded in producing high levels of amorpha-4,11-diene (451) from E. coli [384] and artemisinic acid (473) from S. cerevisiae in quantities which exceed those from A. annua itself (100 mg–1·g·L-1) [385,386].”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [386]
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Cites
The Carbon Monoxide-binding Pigment of Liver Microsomes
Omura, T., & Sato, R. (1964) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(20)82244-3 · Cited by 11,781 · see citing works
Production of the antimalarial drug precursor artemisinic acid in engineered yeast
Ro, D.-K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., et al. (2006) · Nature · doi:10.1038/nature04640 · Cited by 2,805 · see citing works
Self-citationIndustrial biocatalysis today and tomorrow
Schmid, A., Dordick, J. S., Hauer, B., Kiener, A., Wubbolts, M. G., & Witholt, B. (2001) · Nature · doi:10.1038/35051736 · Cited by 2,406 · see citing works
Engineering a mevalonate pathway in Escherichia coli for production of terpenoids
Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J. D., & Keasling, J. D. (2003) · Nature Biotechnology · doi:10.1038/nbt833 · Cited by 1,720 · see citing works
Self-citationSimons, K. T., Kooperberg, C. L., Huang, E. S., & Baker, D. A. (1997) · Journal of Molecular Biology · doi:10.1006/jmbi.1997.0959 · Cited by 1,387 · see citing works
Self-citationENZYME RECRUITMENT IN EVOLUTION OF NEW FUNCTION
Jensen, R. A. (1976) · Annual Review of Microbiology · doi:10.1146/annurev.mi.30.100176.002205 · Cited by 1,108 · see citing works
Dispelling the Myths--Biocatalysis in Industrial Synthesis
Schoemaker, H. E., Mink, D., & Wubbolts, M. G. (2003) · Science · doi:10.1126/science.1079237 · Cited by 838 · see citing works
Enzyme promiscuity: mechanism and applications
Hult, K., & Berglund, P. (2007) · Trends in biotechnology · doi:10.1016/j.tibtech.2007.03.002 · Cited by 637 · see citing works
Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli
Pitera, D. J., Paddon, C. J., Newman, J. D., & Keasling, J. D. (2006) · Metabolic Engineering · doi:10.1016/j.ymben.2006.11.002 · Cited by 440 · see citing works
Self-citationLaboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase
Glieder, A., Farinas, E. T., & Arnold, F. H. (2002) · Nature Biotechnology · doi:10.1038/nbt744 · Cited by 408 · see citing works
Dendrimer-Metalloporphyrins: Synthesis and Catalysis
Bhyrappa, P., Young, J. K., Moore, J. S., & Suslick, K. S. (1996) · Journal of the American Chemical Society · doi:10.1021/ja953474k · Cited by 393 · see citing works
Engineering Escherichia coli for production of functionalized terpenoids using plant P450s
Chang, M. C. Y., Eachus, R. A., Trieu, W., Ro, D.-K., & Keasling, J. D. (2007) · Nature Chemical Biology · doi:10.1038/nchembio875 · Cited by 364 · see citing works
Self-citationNew algorithms and an in silico benchmark for computational enzyme design
Zanghellini, A., Jiang, L., Wollacott, A. M., Cheng, G., Meiler, J., Althoff, E. A., Röthlisberger, D., & Baker, D. A. (2006) · Protein Science · doi:10.1110/ps.062353106 · Cited by 360 · see citing works
Self-citationRoles of key active-site residues in flavocytochrome P450 BM3
Noble, M. A., Miles, C. S., Chapman, S. K., Lysek, D. A., Mackay, A., REID, G. A., Hanzlik, R. P., & Munro, A. W. (1999) · Biochemical Journal · doi:10.1042/bj3390371 · Cited by 270 · see citing works
Newman, J. D., Marshall, J., Chang, M., Nowroozi, F., Paradise, E., Pitera, D., Newman, K. L., & Keasling, J. D. (2006) · Biotechnology and Bioengineering · doi:10.1002/bit.21017 · Cited by 257 · see citing works
Protein engineering of Bacillus megaterium CYP102
Carmichael, A. B., & Wong, L. (2001) · European Journal of Biochemistry · doi:10.1046/j.1432-1327.2001.02212.x · Cited by 246 · see citing works
Bertea, C. M., Freije, J. R., van der Woude, H., Verstappen, F. W., Perk, L., Marquez, V., De Kraker, J.-W., Posthumus, M. A., Jansen, B. J., de Groot, A., et al. (2005) · Planta Medica · doi:10.1055/s-2005-837749 · Cited by 205 · see citing works
Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3
Sowden, R. J., Yasmin, S., Rees, N. H., Bell, S. G., & Wong, L. (2004) · Organic & Biomolecular Chemistry · doi:10.1039/b413068e · Cited by 174 · see citing works
Monoterpene biosynthesis pathway construction in Escherichia coli
Carter, O. A., Peters, R. J., & Croteau, R. (2003) · Phytochemistry · doi:10.1016/s0031-9422(03)00204-8 · Cited by 157 · see citing works
Lim, S.-J., Jung, Y., Shin, H., & Lee, Y. (2002) · Journal of Bioscience and Bioengineering · doi:10.1016/s1389-1723(02)80235-3 · Cited by 146 · see citing works
de Visser, S. P. (2006) · Journal of the American Chemical Society · doi:10.1021/ja065365j · Cited by 142 · see citing works
Hale, V., Keasling, J. D., Renninger, N. S., & Diagana, T. T. (2007) · American Journal of Tropical Medicine and Hygiene · doi:10.4269/ajtmh.2007.77.198 · Cited by 131 · see citing works
Self-citationWallaart, T. E., van Uden, W., Lubberink, H. G. M., Woerdenbag, H. J., Pras, N., & Quax, W. J. (1999) · Journal of Natural Products · doi:10.1021/np980370p · Cited by 129 · see citing works
Covello, P. S. (2008) · Phytochemistry · doi:10.1016/j.phytochem.2008.10.001 · Cited by 127 · see citing works
Kubo, T., Peters, M. W., Meinhold, P., et al. (2005) · Chemistry - A European Journal · doi:10.1002/chem.200500584 · Cited by 127 · see citing works
Rational re‐design of the substrate binding site of flavocytochrome P450 BM3
Ost, T. W. B., Miles, C. S., Murdoch, J., et al. (2000) · FEBS Letters · doi:10.1016/s0014-5793(00)02267-5 · Cited by 118 · see citing works
van Kranenburg, R., Boels, I. C., Kleerebezem, M., & de Vos, W. M. (1999) · Current Opinion in Biotechnology · doi:10.1016/s0958-1669(99)00017-8 · Cited by 107 · see citing works
Thomas, A. F., & BESSIERE, Y. (1989) · Natural Product Reports · doi:10.1039/np9890600291 · Cited by 104 · see citing works
On the conversion of dihydroartemisinic acid into artemisinin
Acton, N., & Roth, R. J. (1992) · The Journal of Organic Chemistry · doi:10.1021/jo00039a020 · Cited by 103 · cited 3× · see citing works
“Dihydroartemisinic acid is the immedi-a te precursor to artemisinin and has been shown to b e readily transformed (8, 9).”
BackgroundLee, W.-H., Park, J., Park, K., Kim, M., & Seo, J. (2007) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-007-1016-7 · Cited by 96 · see citing works
Regio- and stereoselective cleavage of epoxides with cyanoborohydride and boron trifluoride etherate
Hutchins, R. O., Taffer, I. M., & Burgoyne, W. F. (1981) · The Journal of Organic Chemistry · doi:10.1021/jo00338a031 · Cited by 91 · see citing works
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Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development
Paddon, C. J., & Keasling, J. D. (2014) · Nature Reviews Microbiology · doi:10.1038/nrmicro3240 · Cited by 675 · see citing works
Self-citationThe Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao)
Brown, G. D. (2010) · Molecules · doi:10.3390/molecules15117603 · Cited by 363 · see citing works
Zhang, F., Fu, X., Lv, Z., Lu, X., Shen, Q., Zhang, L., Zhu, M., Wang, G., Sun, X., Liao, Z., et al. (2015) · Molecular Plant · doi:10.1016/j.molp.2014.12.004 · Cited by 248 · see citing works
- Authors
- Dietrich, J. A., Yoshikuni, Y., Fisher, K. J., Woolard, F. X., Ockey, D., McPhee, D. J., Renninger, N. S., Chang, M. C. Y., Baker, D., & Keasling, J. D.
- Affiliations
- Lawrence Berkeley National Laboratory (US)
- Howard Hughes Medical Institute (US)
- Amyris (United States) (US)
- QB3 (US)
- Corresponding author
- Keasling, Jay D.
- Venue
- ACS Chemical Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2009-03-20
- Volume
- 4
- Issue
- 4
- Pages
- 261-267
- ISSN
- 1554-8929 (ISSN-L)
- 1554-8937
- OpenAlex
- W2036428713
- Cited by
- 186
- References
- 31
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 4 · 2023: 3 · 2022: 6 · 2021: 6 · 2020: 11 · 2019: 10 · 2018: 11 · 2017: 10 · 2016: 15 · 2015: 18 · 2014: 18 · 2013: 12 · 2012: 19
- FWCI
- 7.66
- Type
- journal article
- Language
- en
- Open access
- green
- www.osti.gov
- Locations
- ACS Chemical Biology (publishedVersion)
- PubMed (publishedVersion)
- Research Explorer (The University of Manchester) (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Keywords
- artemisinin production, cytochrome P450 BM3, active site mutations, dihydroartemisinic acid, Bacillus megaterium, Escherichia coli, heterologous pathway engineering, antimalarial drugs
- MeSH terms
- Polycyclic Sesquiterpenes, Algorithms, Bacillus megaterium, Bacterial Proteins, Computer Simulation, Cytochrome P-450 Enzyme System, Models, Molecular, Molecular Conformation, Mutation, NADPH-Ferrihemoprotein Reductase, Oxidation-Reduction, Sesquiterpenes, Stereoisomerism, Time Factors, Protein Engineering, Crystallography, X-Ray, Catalytic Domain, Artemisinins
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